Multiscan spectrum spectrophotometer

The plasma concentrations of interleukin-1 (IL-1), IL-2, IL-6, IL-10, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), immune globulin A (IgA), (IgG), and IgM were determined using the ELISA assay kits (Huyu, Shanghai, China), according to the manufacturer's protocols. The Multiscan Spectrum Spectrophotometer (Tecan, Infinite M200 Pro, Männedorf, Switzerland) was used to detect the absorbance values.

Approximately 100 mg of frozen jejunum and colon tissues was removed quickly and homogenized with ice-cold physiologic saline (1 : 9, w/v) and then centrifuged at 2,000 g for 20 min at 4°C. The intestinal supernatants were used for further analysis. Plasma and intestinal antioxidant indicators, including catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px), as well as malondialdehyde (MDA) were analyzed by ELISA assay kits from Jiangsu Meimian Institute (Mei mian, Yancheng, China). The total antioxidant capacity (T-AOC) and H₂O₂ assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The test for each index was carried out according to the instructions of the kits. The absorbance values were read on a Multiscan Spectrum Spectrophotometer (Tecan, Infinite M200 Pro, Switzerland). The jejunal and colonic mucous antioxidant parameters were normalized to the total protein concentration (mg/L) quantified by the Pierce BCA Protein Assay Kit (CoWin Biosciences, Suzhou, China).

The ATP concentration of the jejunal and colonic mucosa was determined using the ATP assay kit (Mei mian, Yancheng, China) based on firefly luciferase by a Multiscan Spectrum Spectrophotometer (Tecan, Infinite M200 Pro, Switzerland). The methods for intestinal tissue homogenization and total protein quantification were the same as mentioned above.

Jejunal and ileal mucosa (n = 8) were thawed on ice. Mucosa (1 g) was extracted by adding 9 mL ice-cold PBS (1×), vortexed, and then centrifuged at 4 °C and 3000× g for 15 min. The levels of amylase, chymase, invertase, lactase, lipase, maltase, and trypsin were determined using commercial kits (Shanghai Huyu, Shanghai, China) following the manufacturer’s instructions. A Multiscan Spectrum Spectrophotometer (Tecan, Infinite M200 Pro, Basel, Switzerland) was used for absorbance values. The enzyme activities were normalized to the total protein concentration quantified by the Pierce BCA Protein Assay Kit (Shanghai Huyu, Shanghai, China) and calculated by the total protein unit.

Approximately 0.1 g of frozen liver, ileum, and jejunum tissues were removed quickly, respectively, homogenized with ice-cold physiologic saline (1:9, w/v), and centrifuged at 2,000 × g for 20 min at 4°C. The supernatants were collected for oxidant/antioxidant capacity determination. The liver, ileum, jejunum, and plasma oxidant/antioxidant parameters, including catalase (CAT), glutathione (GSH), H₂O₂, malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) were analyzed by the colorimetric method with the commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The oxidant/antioxidant parameters of liver, jejunum, and ileum tissues were normalized to the total protein concentration quantified by the Pierce BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). The Multiscan Spectrum Spectrophotometer (Tecan, Infinite M200 Pro, Männedorf, Switzerland) was used to detect the absorbance values.

Colonic mucosa samples were prepared as mentioned above. The total protein content of colonic mucous was quantified using the Pierce BCA Protein Assay Kit (CoWin Biosciences, Jiangsu, China). The immunoglobulins and cytokines in the plasma and colonic tissues were measured using the commercially available ELISA kits (Jiangsu Yutong Biological Technology, Jiangsu, China), as per the manufacturer's instructions. The absorbance levels were read on a Multiscan Spectrum Spectrophotometer (Tecan, Infinite M200 Pro, Switzerland). The concentrations of Ig and cytokines in the intestinal mucosa were standardized to the total protein in each sample.

Approximately 100 mg of frozen jejunum and ileum mucosa samples were removed quickly, homogenized with ice-cold PBS (1:9, w/v), and then centrifuged at 2,000 ×g for 20 min at 4°C. The levels of amylase, trypsin, and lipase in the intestinal supernatants were determined using the porcine ELISA kits (Huyu, Shanghai, China), following the manufacturer’s instructions. The absorption values were read on Multiscan Spectrum Spectrophotometer (Tecan, Infinite M200 Pro, Switzerland). The enzyme levels were normalized to the total protein concentration quantified by the Pierce BCA Protein Assay Kit (Beyotime, Shanghai, China).